The development of hepatocellular carcinoma (HCC), the fifth most common cancer worldwide, is largely associated with known risk factors;common risk factors include chronic infections of the liver with the human hepatitis B virus (HBV) and chronic alcohol consumption. In individuals with both risk factors, there is an increase in the presence of HCC. Understanding how chronic HBV infections and alcohol consumption affect the development of HCC and transformation is important in developing therapies for preventing HBV and alcohol-associated HCC. My application focuses on the effect of the nonstructural, hepatitis B virus X protein (HBx) and alcohol exposure on cell proliferation pathways, how exposure to both agents may exacerbate these effects and the consequence for HBV replication and hepatocyte physiology. We have developed methods for isolating and culturing primary rat hepatocytes, thereby generating a biologically relevant system in which to conduct our studies. We will use this system to address the following specific aims: (1) determine the effects of HBx, HBV and ethanol on cell cycle progression by examining cell proliferation and regulation of cell cycle proteins, (2) we will assay the effect of ethanol exposure and cell cycle progression on HBV replication and explore the roles of specific cell cycle proteins in this relationship and (3) determine how ethanol- and HBx dependent regulation of cellular proliferation pathways impacts hepatocyte apoptosis. The long-term goal of these studies is to generate a mechanistic understanding of the effect of HBx and alcohol on cell proliferation in order to create a better understanding of the signaling pathways activated in the development of HCC. We hypothesize that HBx and alcohol regulation of cell cycle progression will ultimately activate apoptotic pathways in hepatocytes and that, when combined, HBx/HBV and alcohol will cause a greater induction of apoptosis. Alcohol consumption and chronic HBV infection cause a high turnover rate of hepatocytes;this may lead to the eventual selection of hepatocytes resistant to apoptotic signals in a regenerating, diseased liver. The combination of a chronic HBV infection and alcohol consumption causes a more rapid progression of liver injury and HCC development than either ethanol exposure or HBV infection alone. The use of normal hepatocytes provides a more biologically relevant system than similar studies performed in immortalized or transformed cells. Our studies will provide a better understanding of the molecular mechanisms contributing to the development of liver cancer.